Chitosan and method of preparing chitosan

ABSTRACT

A highly deacetylated chitosan obtained from microbial biomass, a method of obtaining chitosan from microbial biomass, and biomass for making chitosan are disclosed. The method includes providing chitin-containing biomass; reacting the chitin-containing biomass in a caustic solution of greater than 25 percent alkali at a reaction temperature greater than 95° C. for a reaction period of at least 10 hours to convert the chitin in the biomass to chitosan; and separating the chitosan from the caustic solution.

REFERENCE TO CO-PENDING APPLICATION

This application is a divisional of U.S. application Ser. No.09/739,406, filed Dec. 18, 2000, now allowed, which is acontinuation-in-part of U.S. Provisional Application Ser. No.60/189,560, filed Mar. 15, 2000.

FIELD OF THE INVENTION

The present invention is directed to chitosan and methods of derivingchitosan from chitin-containing biomass.

BACKGROUND

Chitin is a natural polysaccharide present in various marine andterrestrial organisms, including crustacea, insects, mollusks, andmicroorganisms, such as fungi. The structure of chitin is that of anunbranched polymer of 2-acetoamido-2-deoxy-D-glucose(N-acetyl-D-glucosamine), and can be represented by the generalrepeating structure:

Chitin is typically an amorphous solid that is largely insoluble inwater, dilute acids, and alkali. Although chitin has various commercialapplications, greater commercial utility is found by converting chitinto the deacetylated product chitosan. Chitosan can be created byN-deacetylation of the chitin polymer, and its structure may berepresented by the following general formula, wherein at least some ofthe acetylamine groups have been converted to amine groups:

Chitosan is also an amorphous solid that is largely insoluble in water,but is soluble in aqueous organic acids, such as formic and aceticacids. However, the deacetylation reaction is typically not complete,and some of the acetyl groups remain in most chitosan compositions. Inthe representation above, all of the formerly acetylated amine groupshave been converted to amine groups.

Chitosan has many industrial, medical, pharmaceutical, and nutritionaluses, including those requiring a biodegradable, non-toxic polymer. Forexample, chitosan is used as a polyelectrolytic coagulant and a sludgedewatering aid in wastewater treatment. Medical, pharmaceutical, andnutritional uses often require a higher quality chitosan for functionaland aesthetic reasons. These uses include applications asanticoagulants, antiviral agents, drug carriers, cosmetic additives,dialysis membranes, orthopedic materials, wound dressings, foodstabilizers and thickeners, flavor and nutrient carriers, and dietaryfiber.

The quality of chitosan varies with the degree of substitution of theN-acetyl groups, degree of polymerization, manufacturing process, color,clarity, consistency, uniformity, and source. Most chitosan is formed bydissolving calcium carbonate from the shells of aquatic crustacea toliberate chitin, deacetylating the chitin to form chitosan, followed byrecovery and drying of the chitosan. One problem with recovery fromcrustacea is that it is very difficult to obtain uniform, high qualitychitosan. The uniformity problems occur in part because the crustaceatypically are varying sizes, ages, and species; grow under variedenvironmental conditions; and are gathered from different locations. Thequality issues arise in part due to the fact that sufficiently uniformchitosan cannot be obtained, but also include the fact that chitosanobtained from crustacea often has high ash content and can contain heavymetals that is concentrated in the crustacea from their aquaticenvironment. A further problem with chitosan derived from harvestedcrustacea is that it has the potential to include undesired proteins andallergens.

Other methods of producing chitosan involve recovery from microbialbiomass, such as the method taught by U.S. Pat. No. 4,806,474.Unfortunately, existing methods of recovering chitosan from microbialbiomass need improvement to produce higher quality chitosan that is moresuited for pharmaceutical, nutritional and cosmetic applications. Forexample, a need exists for chitosan having improved consistency andsolubility, as well as higher levels of deacetylation than is currentlypracticed. Present processes do not allow for sufficiently high levelsof deacetylation while also providing high quality chitosan from aconsistent and controlled raw material source. For example,deacetylation levels of less than 75 percent can be obtained by methodstaught in U.S. Pat. No. 4,806,474 to Herschberger, but even higherdeacetylation levels are desired. When these higher deacetylation levelsare obtained, it is also desirable that other properties of the chitosanbe retained or improved. Another method, taught by U.S. Pat. No.4,282,351, teaches only how to create a chitosan-beta-glucan complex.

Therefore, a need exists for an improved chitosan material that isobtained utilizing an improved method.

SUMMARY OF THE INVENTION

The present invention is directed to chitosan obtained from microbialbiomass, a method of obtaining chitosan from microbial biomass, andbiomass for making quality chitosan. Chitosan of the present inventiontypically has at least 85 percent deacetylation levels, frequently 90percent deacetylation of the acetyl groups in the chitin, and oftengreater than 95 percent deacetylation. Thus, the compositions typicallyhave an acetylation level of less than 15 percent, frequently less than10 percent, and often less than 5 percent. This level of deacetylationprovides a high quality chitosan with consistent properties that isreadily soluble in a slightly acidic solution.

Chitosan recovered in accordance with the present invention can alsohave improved properties over prior chitosan produced from microbialbiomass as well as from shells from aquatic invertebrates, such asplankton. Some chitosan prepared in accordance with the invention canhave high solubility and low viscosity compared to that which is knownin the art. For example, specific chitosan material prepared inaccordance with the invention may have a viscosity of less than 25centipoise when a 1% solution of the chitosan is dissolved in 1% aqueousacetic acid solution at 25° C.; and may have a viscosity of less than 15centipoise in some implementations under these conditions.

The method of making the chitosan of the present invention includesproviding a consistent chitin-containing biomass; reacting thechitin-containing biomass in a caustic aqueous solution of greater than25 percent alkali at a reaction temperature greater than 95° C. for areaction period of at least 10 hours to convert the chitin in thebiomass to chitosan; and separating the chitosan from the causticsolution. In one implementation of the invention, the chitin-containingbiomass is reacted in a caustic solution of greater than 25 percentalkali at a reaction temperature from 105 to 125° C. for a reactionperiod of 10 to 16 hours to convert the chitin in the biomass tochitosan. More generally, the chitin-containing biomass is typicallyreacted in a caustic solution that is from 30 to 40 percent alkali.Suitable reaction temperatures for reacting the biomass are generallyless than 125° C., and reaction periods are generally from 10 to 20hours, and typically from 10 to 16 hours. The method of obtainingchitosan from microbial biomass may also include washing thedeacetylated biomass with a caustic solution, recovery of the chitosan,precipitating the chitosan, and drying the precipitated chitosan.

In addition to the primary deacetylation reaction, a pre-treating stepmay also be used in which the microbial biomass is heated in a lessalkaline solution prior to reacting it in a more alkaline solution todeacetylate the chitin. Typically the alkaline concentration isinitially below 10 percent for this pre-treatment step and subsequentlyraised to greater than 25 percent for the primary reaction. In specificpre-treatment implementations, the biomass is first heated in a causticconcentration of about 2 to 5 percent alkali for 0.5 to 4.0 hours at atemperature of 100 to 120° C. This pre-treatment aids in removing excessproteins and various contaminants to provide a higher quality chitosan.

The chitosan is prepared from chitin contained in microbial biomass, andin particular fungal biomass. Suitable microbial biomasses includeAspergillus niger, Aspergillus terreus, Aspergillus oryzae, Lactariusvellereus, Mucor rouxii, Penicillium chrysogenum, Penicillium notatum,Saccharomyces cerevisiae; and in particular Candida guillermondi,Aspergillus niger, and Aspergillus terreus. Preferably, the biomass isrecovered from a commercial fermentation reaction, such as thecommercial production of organic acids, such as citric acid. As useherein, the term microbial does not include phyto-plankton andcrustaceans or mollusks.

The above summary of the present invention is not intended to describeeach disclosed embodiment of the present invention. This is the purposeof the detailed description and claims which follow.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to chitosan obtained from microbialbiomass, to a method of obtaining chitosan from microbial biomass, andto biomass for making chitosan. The chitosan is typically derived fromsubstantially uniform fungal sources, and has superior propertiesrelative to known products. The method includes providingchitin-containing biomass; reacting the chitin-containing biomass in acaustic solution of greater than 25 percent alkali at a reactiontemperature greater than 95° C. for a reaction period of at least 10hours to convert the chitin to chitosan; and separating the chitosanfrom the caustic solution.

Specific aspects of the chitosan, biomass, and method of the inventionare described below.

Chitosan

Chitosan as produced with the present invention has improved propertiesover prior chitosan produced from microbial biomass. Some chitosanprepared in accordance with the invention has high solubility and lowviscosity compared to that which is known in the art. For example, somechitosan material of the present invention has a viscosity of less than25 centipoise when dissolved in a 1% solution of the separated chitosanin 1% acetic acid; and may have a viscosity of less than 15 centipoisein some implementations.

Chitosan of the present invention typically has at least 85 percentdeacetylation of the acetyl groups in the chitin, frequently at least 90percent deacetylation, and often greater than 95 percent deacetylation.These levels of deacetylation provides a high quality chitosan that isreadily soluble in slightly acidic solution. Notably, thesedeacetylation levels are obtained without excessive damage to thechitosan molecule. Thus, the molecule can maintain its integrity whileimproving performance.

In addition, the chitosan of the present invention typically has greateruniformity than that observed in prior chitosan. This uniformityincludes, for example, viscosity, color, and deacetylation levels. Anadditional improvement of the present chitosan is that it has low ashlevels, typically less than 0.50 percent. Also, the chitosan has lowlevels of heavy metals, particularly in comparison to prior art chitosanproduced from crustacea, and is typically less than 1.0 parts permillion and preferably less than 0.5 parts per million. The improvedcolor uniformity, lack of ash, and low heavy metals levels havesignificant advantages, depending upon the application. For example, forcosmetic purposes color uniformity and clarity of the chitosan can bevery desirable. Similarly, the lack of ash results in improved clarityand purity, thus providing advantages for use in both medical anddietary applications. Low heavy metals levels also provide significantadvantage when the chitosan is used for medical or dietary purposes.

Chitin-Containing Biomass

The present invention is directed to chitosan recovered from microbialbiomass, in particular fungal biomass, including yeast and filamentousfungi. Suitable microbial biomass may be obtained from Aspergillusniger, Aspergillus terreus, Aspergillus oryzae, Candida guillermondii,Lactarius vellereus, Mucor rouxii, Penicillium chrysogenum, Penicilliumnotatum, Saccharomyces cerevisiae; and in particular CandidaGuillermondii, Aspergillus niger, or Aspergillus terreus. Although it ispossible to generate biomass solely for the purpose of obtainingchitosan, the biomass most often is a by-product of other productionprocesses. For example, citric-acid fermentation facilities use fungi tocreate citric acid. Traditionally, the fungal biomass from citric-acidfermentation has been discarded or used as fuel, feed, or fertilizer.However, the present invention allows extraction of high qualitychitosan from this fungal biomass.

Biomass suitable for use in the present invention includes most types ofchitin-containing microbial biomass, and in particular fungal biomass.The invention is particularly well suited to uses where the chitinlevels exceed 5 percent of the dry biomass weight. Such biomass usuallyhas between 5 and 25 percent chitin, and typically from 10 to 20 percentchitin, based upon dry weight of the biomass. In order to prepare thehighest quality chitosan, it is sometimes desirable that the microbialbiomass be produced in a substantially controlled manner havingrelatively uniform temperature and nutrient levels during the growth ofthe biomass.

Method of Obtaining Chitosan from Chitin-Containing Fungal Biomass

The present invention is also directed, in part, to improved methods ofproducing chitosan from chitin-containing biomass. The chitosan producedby the improved methods shows desirable properties which make thechitosan well-suited for various applications. These properties include,in certain implementations, favorable levels of deacetylation,viscosity, color, and/or ash.

In general, the method comprises reacting chitin-containing biomass in acaustic solution followed by recovery of the chitosan from the solution.In one embodiment of the invention, the chitin-containing biomass is afungal biomass reacted in a caustic solution of greater than 25 percentalkali at a reaction temperature from 105 to 125° C. for a reactionperiod of 10 to 16 hours to convert the chitin in the biomass tochitosan. More generally, the chitin-containing biomass is typicallyreacted in a caustic solution that is from 30 to 40 percent alkali.Suitable reaction temperatures for reacting the biomass are generallyless than 125° C., and reaction periods are generally from 10 to 20hours, and typically from 10 to 16 hours.

The method of obtaining chitosan from microbial biomass may also includewashing the deacetylated biomass with a caustic solution; recovery ofthe chitosan; precipitating the chitosan, and drying the precipitatedchitosan. A pre-treating step may also be used in which the microbialbiomass is heated at with a caustic solution with a lower percent alkaliprior to reacting it at a higher caustic solution. Typically the causticconcentration is initially below 10 percent and subsequently raised togreater than 25 percent during the pre-washing step.

Chitosan Uses

The improved chitosan can be advantageously used in variousapplications. These applications include pharmaceuticals, generalmedical uses, textiles and films, nutritional and dietary products,filtration products and methods, and various other industrial andconsumer products and processes. Specific products and uses are brieflydescribed below.

With regard to pharmaceuticals, the chitosan can be used in orallyadministrable pharmaceutical compositions, their derivatives, and saltsthereof; in drug capsules; as a carrier for various pharmaceuticals,including anticancer agents; as a bone growth matrix and in variousdental applications; and to reduce blood cholesterol levels.

The chitosan is also suitable for use in general medical applications,such as to make dressings made of cotton-like chitosan fibers, includingnon-woven, biodegradable wound and burn dressings; as a treatment forskin ulcerations; to make temporary artificial ligaments; to promotetissue regeneration; in swellable wound dressings; as encapsulatingagents for cell transplantation; as a soft tissue, skin or bone growthmatrix; and as wound-healing ointments, sutures, artificial ligaments,etc.

The chitosan is suitable for use in food and dietary products; includingin beverages, in diet foods, and in health foods. The chitosan can beused as stabilizers, packaging films, preservative coatings, cholesteroland fat-reducing agents (including as a fat binding agent for dietaryand non-dietary applications), and chewing gums. In addition, thechitosan can be used as a fining agent to clarify beverages; and to aidin the retention of post-cooked flavor in beef by binding to the iron inthe beef and maintaining its flavor potential.

The chitosan can be used in various textile and film applications. Theseapplications include use in anti-bacterial, mildew-proof and deodorantfibers for making underwear, socks, bed sheets for hospitals, bandages,diapers, pillow covers, gloves, towels, aprons, and bedding; andspecialized cleaning absorbents, such as synthetic absorbent sponges.The chitosan can be incorporated into fabric for wrapping material forfoods, tea bags, disposable warmers, etc; and into antibacterial andfreshness-preserving non-woven fabrics. In specific implementations thechitosan can be used as a coating on various fabrics, including rayon.The chitosan is suitable for use in water-permeable fabrics for sportsand professional use, and methods for facilitating cloth production.

The improved chitosan can be incorporated and used in various cosmeticproducts. For example, the chitosan may be incorporated into hairshampoos, rinses, perming agents, colorants, sprays and tonics, skincreams and lotions, eye shadows, lipsticks, foundations, nail enamels,and other cosmetics, toothpastes and mouthwashes. The chitosan can beused in deodorant formulas to inhibit bacterial growth on the skinsurface, thereby preventing perspiration odor. In hair styling products,chitosan adds desired form and strength, while preventing the hair fromdrying out or splitting. Also, the chitosan can be used in treatment of“orange peel skin” because of its ability to form a clear protectivecoating possessing moisture retention properties and being non-shellfishallergenic. In hair care products chitosan can form films with hairkeratin. The chitosan can be used in pigments in color cosmetics; andcan be used to coat dispersed pigment particles and soften theconsistency of such pigmented cosmetics.

The chitosan has numerous uses in agriculture, including use inbiodegradable films based on cellulose, starch and chitosan, may bedecomposed by soil microbes, and can be used in agricultural andfisheries applications. Chitosan tends to increase root and stem growthand is can be effective in creating immunity to disease in plants.Furthermore, the chitosan can be used as a coating for delayed ripeningof fruit, and as a seed treatment. The chitosan film has good wet anddry strength, and is suitable for agricultural applications such astapes for seeds and spore bags for aver farming.

Various water treatment and filtration processes are amenable to usewith the improved chitosan, such as use as micro porous or macro porousaffinity filtration membranes for removal of metals, pesticides andPCBs. The chitosan can be used as for producing solid supports forvarious chromatographic methods, chelating agents, adsorbents, carriesfor enzymes or cells, and time-release bases for physiologically activesubstances; and can be used as treatments for organic (such asbioremediation) or heavy-metal pollutants and clarifiers for pools,spas, or natural bodies of water, as well as waste recovery.Specifically, chelating-based recovery systems for metals used invarious industries, including electroplating, electronics manufacturing,metal finishing, photo processing and jewelry production may be usedthat contain chitosan. Chitosan adsorbents can be effective for removalof radio nuclides, removal of metals, pesticides and PCBs from liquids.In particular implementations the chitosan may be used as treatments fororganic or heavy-metal pollutants and clarifiers for pools, spas, ornatural bodies of water. Finally the chitosan can be used as a sewagetreatment additive and chelating agent for removal of traces of heavymetals from aqueous solutions.

Numerous industrial applications are appropriate for use with theimproved chitosan, including use in adhesives and as a coating toimprove dyeing characteristics of glass fibers. Further, the improvedchitosan can be used as a wet strength additive for paper, adhesives,photographic and printing applications, and can act as a ‘retention aid’in paper. Specific paper applications include use as a retention agentin the manufacture of paper. As such, it acts to retain titaniumdioxide, ash components, calcium carbonate, silica derivatives, and pulpfines. Chitosan can also be used as a printing aid to help withinrunability. The chitosan can help retain components such as ash(opacity, whitening agents) and fiber fines in the pulp mixture as it isbeing pressed into paper. The chitosan can also be used to improve thestrength of a final paper product, which helps decrease potential downtime during processing.

In addition, various other applications and uses for the chitosan arealso appropriate.

Example Method of Obtaining Chitosan from Chitin-Containing Biomass

The following example is provided to demonstrate recovery of chitosanfrom chitin in accordance with an implementation of the invention. Inthe example depicted, the chitosan was recovered under laboratoryconditions. However, the invention is especially applicable toproduction of chitosan in large-scale manufacturing operations whereparticularly uniform sources of fungal biomass may be obtained.

250 grams of microbial biomass of Aspergillus niger was mixed withapproximately 250 mL of a 4 percent by weight aqueous sodium hydroxidesolution. The alkali biomass solution was heated in an autoclave for 30minutes at a temperature of 120° C. in order to pre-treat the biomassand aid in the removal of proteins, lipids, and various coloredimpurities. After heating, the warm solution was filtered, and thesolids washed with deionized water until the water ran substantiallyclear. This pre-treated biomass can be immediately processed todeacetylate the chitin, but can optionally be stored.

For this example, approximately 200 grams of pre-treated biomass, whichoften contains 80 to 86 percent water, was placed in a polypropylenebottle on a top load balance, followed by addition of 180 grams of 50percent aqueous solution of sodium hydroxide, and 60 grams of sodiumhydroxide pellets. The reaction mixture typically contains 32 to 38percent sodium hydroxide, 55 to 65 percent water, and 3 to 9 percent drypre-treated biomass.

A strongly exothermic reaction occurred, and the sodium hydroxidepellets dissolved into solution with the biomass and water. Thepolypropylene bottle containing the biomass in the alkali sodiumhydroxide solution was placed in a preheated oven at approximately 120°C. The bottle was loosely covered in order to allow escape of gases.

The solution was maintained at a temperature of 120° C. for 16 hours,after which it was removed from the oven and 200 mL of near-boilingwater was gently added to the solution. This hot mixture was filteredthrough filter cloth, and the solids rinsed with additional hotdeionized water. The solids, containing a chitosan-glucan complex, wererinsed with approximately 500 mL of water approximately 10 times untilthe pH of the filtered solution was below 9.

After rinsing, the solids were transferred to a beaker and glacialacetic acid was added until a pH of between 3.5 and 5.0 was obtained.The mixture was stirred for approximately 10 minutes to extract chitosanfrom the glucan, and the mixture centrifuged. The supernatant wascollected in an Erlenmeyer flask Optionally, the residue in thecentrifuge container may be rinsed with a slightly acidic solution toremove any additional chitosan and re-centrifuged. The supernatantcontaining dissolved chitosan from the two centrifuge steps may then becombined and further processed.

The solution from the supernatant was moderately turbid, and wasfiltered through a 1.5 μm membrane filter followed by filtration througha 0.7 μm membrane filter. The filtration through the 0.7 Tm filter wasrepeated until no visible suspension was observed in the solution. Afterfiltration, a solution of 10 percent sodium hydroxide was added to thefiltered chitosan-containing solution, until a pH of between 10.5 and11.5 was obtained, at which time a white solid containing chitosanprecipitated from the solution. The mixture was set aside for 10 minutesuntil the precipitate separated out of the solution. The mixture wasdecanted, followed by rinsing with water to remove excess sodiumhydroxide. The basic mixture was placed in a centrifuge bottle, whichwas centrifuged and decanted. Additional deionized water was added tothe centrifuge residue, which was shaken to suspend all solids,centrifuged again, and decanted. These steps of centrifuging, decanting,and washing were repeated until the pH dropped below 9.0. Care was takento reduce the time that the chitosan at excessively high or low pH forlong periods of time to avoid depolymerization or degradation. Theresulting residue was freeze dried.

The recovered chitosan had the following characteristics: viscosity(centipoise)   1 to 10 turbidity   5 to 20 NTU ash percent 0.20 to 0.40color  120 to 170 APHA deacetylation   92 to 97 percent

Viscosity was measured using a Brookfield DV-II viscometer, whichmeasures fluid resistance using a moving pendulum in motion. A solutionof 1 percent chitosan and 1 percent acetic acid in water was preparedand mixed until all chitosan was in solution. The viscometer controlswere set for a large spindle and a temperature of 25° C., after which a16 ml sample was placed within the sample tube. This sample tube waspositioned in the mounting channel of the viscometer, and the viscositymeasured after approximately 5 seconds.

Turbidity was measured using a HACK Ratio/XR turbidometer on a sample of1 percent by weight chitosan (based on dry weight) in 1 percent byweight aqueous acetic acid.

The ash content was measured using a muffle furnace and hot plate. Themuffle furnace was heated to 550° C. (within a range of 25 degrees), anda sample of chitosan was added to a crucible of a known weight. Thissample and crucible were heated in the muffle furnace for 10 hours untilashing was complete, after which the crucible was placed in a desiccatorto cool for 60 minutes. After this cooling period, the weight of thecrucible was measured, and the percent ash calculated using thefollowing formula:% Ash=((Final Crucible Weight)−(Initial Crucible Weight))/(SampleWeight)×100

Color was measured using an Aquatester by Orbeco-Hellige on a sample of1 percent by weight chitosan (based on dry weight) in 1 percent byweight aqueous acetic acid.

Deacetylation was measured using nuclear magnetic resonance (NMR)spectroscopy. Pre-samples were prepared over a period of about 2 to 3hours. To prepare the pre-samples, approximately 50 milligrams ofchitosan-containing material was mixed in de-ionized water (5 mL) in a10 mL test tube. Approximately 1 to 2 drops of formic acid were added tothe sample to dissolve solids, after which the test tube was vigorouslyshaken to dissolve the chitosan. After the chitosan was dissolved,approximately 10 percent sodium hydroxide was added to precipitate thechitosan. The solid was centrifuged and the supernatant decanted.Approximately 1 ml of deionized water was added to the residue. The tubewas vigorously shaken to mix the solid residue into the solution, andthe solution was centrifuged. The shaking and centrifuging steps wererepeated until the supernatant pH was near neutral, at which point 5 mLof water was added to the collected solid, and small amounts of formicacid were added to completely dissolve the solid. These steps wererepeated. The obtained solution was frozen and freeze-dried.

Samples were prepared by measuring approximately 15 to 20 milligrams ofdried sample into a small vial. About 1.5 milliliters of deuteriumhydroxide (D₂O) was added to the sample, which was shaken vigorously todissolve completely, and the NMR spectrum analyzed and used to determinethe degree of deacetylation.

The above specification, examples and data provide a description of thecompositions and methods of the invention. Since many embodiments of theinvention can be made without departing from the spirit and scope of theinvention, the invention resides in the following claims.

1. A method of obtaining chitosan from microbial biomass, the methodcomprising the steps of: (a) providing chitin-containing biomass; (b)reacting the chitin-containing biomass in a caustic solution of greaterthan 25 percent alkali at a reaction temperature greater than 95° C. fora reaction period of at least 10 hours to convert the chitin in thebiomass to chitosan; and (c) separating the chitosan from the causticsolution.
 2. The method of claim 1, wherein the caustic solution is from30 to 40 percent alkali.
 3. The method of claim 1, wherein the reactiontemperature is from 105° to 125° C.
 4. The method of claim 1, whereinthe reaction temperature is less than 125° C.
 5. The method of claim 1,wherein the reaction period is from 10 to 20 hours.
 6. The method ofclaim 1, wherein 1 percent solution of the separated chitosan in 1percent acetic acid has a viscosity of less 25 centipoise at 25° C. 7.The method of claim 1, wherein the chitin is at least 85 percentdeacetylated.
 8. The method of claim 1, wherein the chitin is at least90 percent deacetylated.
 9. The method of claim 1, wherein the chitin isat least 95 percent deacetylated.
 10. The method of claim 1, whereinseparating the chitosan comprises washing the deacetylated biomass witha caustic solution; recovery of the chitosan; precipitating thechitosan, and drying the precipitated chitosan.
 11. The method accordingto claim 1, wherein the microbial biomass is selected from the groupconsisting of Candida Guillermiondii, Aspergillus niger, Aspergillusterreus, and combinations thereof.
 12. The method according to claim 1,further comprising heating the microbial biomass at a causticconcentration off less than 25 percent alkali prior to reacting it at acaustic solution of greater than 25 percent alkali.
 13. The methodaccording to claim 12, wherein the caustic concentration is less than 10percent alkali.
 14. The method according to claim 12, wherein heatingthe microbial biomass is at caustic concentration is from 2 to 5 percentalkali for less than 4 hours at a temperature of 100° to 120° C.